Synthesis, characterization and pharmacological studies of some substituted fluoroquinolones

Article abstract of DOI:10.13005/ojc/290231

Certain N-1 substituted fluoroquinolonic derivatives were synthesized and evaluated for antimicrobial and antioxidant activities. N-1 alkyl/ aryl/ aryl sulphonyl substituted derivatives of the title compounds have been synthesized to identify newer fluoroquinolones which have better efficacy, lesser side effects and well tolerability. The biological evaluation of the synthesized fluoroquinolone derivatives was carried out using agar-well diffusion method and compounds FI-ETH, FII-SUL, FIII-ATY, FIV-BZO, and FV-BZY were found to be active against both Gram-positive and Gram-negative bacteria having activity comparable to that of standard drug i.e. Ofloxacin 10mg/ml. N-1 substituted moiety is mostly active against strain is S. aureus, K. Pneumonia, and E. coli with concentration of 100 - 150μg/ml, and less active against strain is B. subtilis. And secondly, antioxidant activity is that of show the better activity by this four method DPPH Free Radical Scavenging Assay, Hydrogen Peroxide Radical Scavenging Activity, Nitric Oxide Assay, and Reducing Power Assay. All the synthesized compounds show the better antioxidant activity and in fourth one method result is mostly capable reducing power activity of the synthesized compounds.

Full text of DOI:10.13005/ojc/290231

ISSN: 0970-020 X
CODEN: OJCHEG
2013, Vol. 29, No. (2):
Pg. 609-615
ORIENTAL JOURNAL OF CHEMISTRY
An International Open Free Access, Peer Reviewed Research Journal
www.orientjchem.org
Est. 1984
Synthesis, Characterization and Pharmacological
Studies of Some Substituted Fluoroquinolones
FAIZ MOHD. ABDULLAH*and ARVIND KUMAR SINGH
*Kamla Nehru Institute of Management & Technology,
(Faculty of Pharmacy), Faridipur, Sultanpur - 228 118, India.
*Correspondence author E-mail: fmabd2006@yahoo.com
(Received: March 25, 2013; Accepted: April 30, 2013)
ABSTRACT
Certain N-1 substituted fluoroquinolonic derivatives were synthesized and evaluated for
antimicrobial and antioxidant activities. N-1 alkyl/ aryl/ aryl sulphonyl substituted derivatives of the
title compounds have been synthesized to identify newer fluoroquinolones which have better
efficacy, lesser side effects and well tolerability. The biological evaluation of the synthesized
fluoroquinolone derivatives was carried out using agar-well diffusion method and compounds FI-
ETH, FII-SUL, FIII-ATY, FIV-BZO, and FV-BZY were found to be active against both Gram-
positive and Gram-negative bacteria having activity comparable to that of standard drug i.e.
Ofloxacin 10mg/ml. N-1 substituted moiety is mostly active against strain is S. aureus, K. Pneumonia,
and E. coli with concentration of 100 - 150µg/ml, and less active against strain is B. subtilis. And
secondly, antioxidant activity is that of show the better activity by this four method DPPH Free
Radical Scavenging Assay, Hydrogen Peroxide Radical Scavenging Activity, Nitric Oxide Assay,
and Reducing Power Assay. All the synthesized compounds show the better antioxidant activity
and in fourth one method result is mostly capable reducing power activity of the synthesized
compounds.
Key words: Fluoroquinolones, Antibacterials, Antioxidant agent.
INTRODUCTION
fluoroquinolones have been patented and are still
used today, including norfloxacin (1978), pefloxacin
(1979), enoxacin (1980), fleroxacin (1981),
ciprofloxacin (1981) and ofloxacin (1982)1. An
advantage of these compounds over previous ones
is their broad spectrum. A big revolution was made
in 1980 ís when an analog of naldixic acid, enoxacin
was derived with significantly increased spectrum
of activity against Gram negative or Gram positive
bacteria8. An antioxidant is a molecule capable of
inhibiting or preventing the oxidation of other
molecules. Antioxidants are substances that may
The first clinically useful quinolone was
naldixic acid, discovered by Lesher and co-workers
in 1962, which was generated from chloroquine,
an antimalarial agent1. It was active against some
Gram negative bacteria and had limited usefulness
because of its high protein binding (approximately
90%) and little half life (about 1.5 h)6. Unfortunately,
bacteria could develop a rapid resistance to this
agent7,11. Flumequine was the first fluoroquinolone
which was patented in 1973, after that many

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ABDULLAH & SINGH, Orient. J. Chem., Vol. 29(2), 609-615 (2013)
protect cells from the damaging effects of oxygen following method. ethyl 7-chloro-6-fluoro-4-oxo1, 4-
radicals, highly reactive chemicals that play a part dihydroquinoloine-3-carboxylate (iv) 0.01 mol was
in atherosclerosis, some forms of cancer and added to 10 ml. of DMF, followed by addition of N-1
reperfusion injuries (4). Free radicals are created position (R) (0.01 mol). The reaction mixture was
when cells use oxygen to generate energy. These heated to dissolve the 3-acid which was partially
by-products are generally reactive oxygen species soluble in cold condition, and then anhydrous
(ROS) such as super oxide anion, hydroxyl radical potassium carbonate (0.02 mol) was added to the
and hydrogen peroxide that result from the cellular reaction mixture. The whole reaction mixture was
redox process. At low or moderate concentrations, heated to 1200 – 1400C and stirred for 5-8 hrs. then,
(ROS) exert beneficial effects on cellular responses the reaction mixture was poured onto the crushed
and immune function but at high levels, free radicals ice or ice cold water, washed with cold water to
and oxidants generate oxidative stress, a remove DMF and potassium carbonate if any, the
deleterious process that can damage cell structures, solid (v) obtained was recrystillised from acetone to
including lipids, proteins, and DNA (9). Oxidative get the derivative. Showed in scheme 2 fig. II. All
stress plays a major part in the development of synthesized derivatives checked by TLC technique
chronic and degenerative ailments such as cancer, using adsorbent silica gel G of Merck specialties
autoimmune disorders, rheumatoid arthritis, Pvt. Ltd., Mumbai and each derivatives are isolate
cataract,
aging,
cardiovascular
and & purified by column chromatography using silica
neurodegenerative diseases16, 9-10. The human body gel mesh size 60-120 of Merck specialties Pvt. Ltd.,
has several mechanisms to counteract oxidative Mumbai.
stress by producing antioxidants, which are either
naturally produced in situ, or externally supplied In-vitro antibacterial activity (5)
through foods and/or supplements. These
Agar- Well Diffusion Method: Petriplates
antioxidants act as free radical scavengers by containing 20 ml Muller Hinton medium were
preventing and repairing damages caused by ROS, seeded with 24 hr culture of bacterial strains. Wells
and therefore can enhance the immune defense were cut and 20 µl of the given sample (of different
and lower the risk of cancer and degenerative concentrations) were added. The plates were then
diseases9. In recent years, there is an increasing incubated at 37°C for 24 hours. The antibacterial
interest in finding antioxidant phytochemicals, activity was assayed by measuring the diameter of
because they can inhibit the propagation of free the inhibition zone formed around the well. Ofloxacin
radical reactions, protect the human body from were used as a positive control.
diseases12-16
.
In-vitro antioxidant activity by
Methodology
Synthesis of derivative FI-ETH Equimolar
DPPH Free Radical Scavenging Assay (2, 10)
The stable 1,1-diphenyl-2-picryl hydrazyl
amounts of 3-chloro-4-fluoroaniline (i), and radical (DPPH) was used for determination of free
diethylethoxymethylene malonate (ii) were radical-scavenging activity of the sample Different
condensed at 1450C to get 3-chloro-4- concentrations (50 – 250 µg/ml) of each sample
fluoroanilinomethylene malonic diethyl ester (iii), were added, at an equal volume, to 90% methanolic
which was then cyclized by heating at 2500C in solution of DPPH (100 µM). After 15 min at room
diphenyl ether to get ethyl 7-chloro-6-fluoro-4-oxo1, temperature, the absorbance was recorded at 517
4-dihydroquinoloine-3-carboxylate (iv). Whole nm. The experiment was repeated for three times.
reaction mixture change into the semisolid mass Ascorbic acid were used as standard controls. IC50
having white to pale yellow appearance and values denote the concentration of sample, which
washed with acetone to get almost white solid is required to scavenge 50% of DPPH free radicals.
recrystillised using DMF as solvent. Showed in
scheme 1 fig. I.
Hydrogen Peroxide Radical Scavenging Activity
(4)
Synthesized derivatives (FII-SUL, FIII-ATY,
The ability of the sample (FI-ETH, FII-SUL,
FIV-BZO, and FV-BZY) are synthesized by this FIII-ATY, FIV-BZO, and FV-BZY) to scavenge

ABDULLAH & SINGH, Orient. J. Chem., Vol. 29(2), 609-615 (2013)
611
hydrogen peroxide was determined. A solution of
hydrogen peroxide (40 mM) was prepared in
phosphate buffer (pH 7.4). The concentration of
hydrogen peroxide was determined by absorption
at 230 nm using a spectrophotometer. Sample (50
– 250 µg/ml) in distilled water, and 1 ml added to
2.4 ml. of 0.1 M phosphate buffer solution and a
hydrogen peroxide solution (600 µl, 40 mM). The
absorbance of hydrogen peroxide at 230 nm was
determined after 10 minutes against a blank
solution containing phosphate buffer without
hydrogen peroxide. The percentage of hydrogen
peroxide scavenging by the extracts and standard
compounds was calculated as follows: %
Scavenged [H2O2] = [(Ao - A1)/Ao] × 100 where Ao
was the absorbance of the control and A1 was the
absorbance in the presence of the sample and
standard.
distilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1%),
and the absorbance was measured at 700 nm.
Increased absorbance of the reaction mixture
indicated increased reducing power. Ascorbic acid
used as a standard.
Spectral Analysis
All synthesized derivatives done
characterized by 1H NMR, MASS, and FT-IR.
FI-ETH: FT-IR (cm-1)
1445 (C-N stretch of quinolines having
C=C-N), 1044 (C-F), 751 (C-Cl), 1650 (C=C-C=O),
1733 (C=C-COOR), 1618 (N-H bending).
MS (m/z)
269 (M+) C12H9ClFNO3 calculated value:
C= 53.53, H= 3.35, Cl= 13.20, F= 7.05, N= 5.2, O=
17.84; 270 (M+) Found value: C= 53.33, H= 3.33,
Cl= 13.14, F= 7.03, N= 5.18, O= 17.77.
Nitric Oxide Assay (13)
The procedure is based on the principle
that, sodium nitroprusside in aqueous solution at
physiological pH spontaneously generates nitric
oxide which interacts with oxygen to produce nitrite
ions that can be estimated using Griess reagent.
Scavengers of nitric oxide compete with oxygen,
leading to reduced production of nitrite ions. For
the experiment, sodium nitroprusside (5 mM), in
phosphate-buffered saline, was mixed with different
concentrations (50 - 250 µg/ml) of each sample
dissolved in methanol and incubated at room
temperature for 30 min. After the incubation period,
1.5 ml incubated solution was removed and diluted
with 1.5 ml of Griess reagent (1% sulphanilamide,
2% phosphoric acid and 0.1% N-1-
naphthyethylenediamine dihydrochloride) was
added.The absorbance of the chromophore formed
was read at 546 nm. Rutin was used as standard.
+H NMR (dmso d6 300 MHz) δ ppm= 7.06
(1H, s, J= 7.021), 7.46 (1H, s, J= 7.424), 4.15 (1H, s,
J= 4.133), 1.13 (3H, m, J= 1.056), 4.4 (2H, m, J=
4.353), 8.0 (1H, s, J= 7.954).
FII-SUL: FT-IR (cm-1)
1450 (C-N stretch of quinolines C=C-N),
1044 (C-F), 637 (C-Cl), 1635 (C=C-C=O), 1733
(C=C-COOR), 3325 (aromatic C-H), 2972 (C-H
alkane), 1380 (SO2 Symmetric), 1274 (SO2
Asymmetric).
MS (m/z)
423 (M+), C19H15ClFNO5S calculated value:
C= 53.90, H= 3.54, Cl= 8.39, F= 4.49, N= 3.30, O=
18.91, S= 7.5; 422 (M+) Found value: C= 53.77, H=
3.53, Cl= 8.37, F= 4.48, N= 3.30, O= 18.86, S= 7.56.
Reducing Power Assay (3)
+H NMR (dmso d6 300 MHz) δ ppm= 7.06
(1H, s, J= 7.021), 7.46 (1H, s, J= 7.424), 1.13 (3H,
m, J= 1.056), 4.4 (2H, m, J= 4.353), 8.0 (1H, s, J=
7.954), 7.3 (2H, m, J= 7.414), 7.71 (2H, m, J= 7.734),
2.28 (3H, m, J= 2.281).
The reducing power assay of sample was
determined. Different amounts of each sample (50
-800 µg/ ml) in 1 ml of methanol were mixed with
phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and
potassium ferrocyanide [K3Fe(CN)6] (2.5 ml, 1%).
The mixture was incubated at 50oC for 20 min. A
portion (2.5 ml) of trichloroacetic acid (10%) was
added to the mixture to stop the reaction, which
was then centrifuged at 3000 rpm for 10 min. The
upper layer of solution (2.5 ml) was mixed with
FIII-ATY: FT-IR (cm-1)
1425 (C-N stretch of quinolines C=C-N),
1044 (C-F), 659 (C-Cl), 1649 (C=C-C=O), 1733
(C=C-COOR), 2973 (C-H alkane).

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ABDULLAH & SINGH, Orient. J. Chem., Vol. 29(2), 609-615 (2013)
MS (m/z)
+H NMR (dmso d6 300 MHz) δ ppm= 7.83
311 (M+), C14H11ClFNO4 calculated value: (1H, m, J= 7.844), 7.54 (1H, m, J= 7.615), 8.75 (1H,
C= 54.01, H= 3.5, Cl= 11.41, F= 6.1, N= 4.5, O= s, J= 8.728), 1.13 (3H, m, J= 1.056), 4.4 (2H, m, J=
20.5; 311 (M+) Found value: C= 54.01, H= 3.5, Cl= 4.353), 1.13 (3H, m, J= 1.056), 4.4 (2H, m, J= 4.353),
11.41, F= 6.1, N= 4.5, O= 20.5.
7.79 (2H, m, J= 7.812), 7.34 (2H, m, J= 7.386), 7.57
(1H, s, J= 7.601).
+H NMR (dmso d6 300 MHz) δ ppm= 7.83
(1H, m, J= 7.844), 7.54 (1H, m, J= 7.615), 8.75 (1H, FV-BZY: FT-IR (cm-1)
s, J= 8.728), 1.13 (3H, m, J= 1.056), 4.4 (2H, m, J=
4.353), 2.27 (3H, s, J= 2.299).
1494 (C-N stretch quinolines having C=C-
N), 881 (C-F), 658 (C-Cl), 1655 (C=C-C=O), 3423
(Aromatic C-H), 2969 (C-H alkane).
FIV-BZO: FT-IR (cm-1)
1466 (C-N stretch of quinolines C=C-N), MS (m/z)
1030 (C-F), 618 (C-Cl), 1690 (C=C-C=O), 3099
359 (M+) C19H15ClFNO3 calculated value:
(aromatic C-H).
C= 63.50, H= 4.17, Cl= 9.88, F= 5.29, N= 3.89, O=
13.37; 359 (M+) Found value: C= 63.50, H= 4.17,
Cl= 9.88, F= 5.29, N= 3.89, O= 13.37.
MS (m/z)
373 (M+) C19H13ClFNO3 calculated value:
C= 61.12, H= 3.48, Cl= 9.51, F= 5.09, N= 3.75, O=
17.15; 373 (M+) Found value: C= 61.12, H= 3.48, (1H, s, J= 7.299), 7.97 (1H, m, J= 7.973), 4.26 (2H,
+H NMR (dmso d6 300 MHz) δ ppm= 7.23
Cl= 9.51, F= 5.09, N= 3.75, O= 17.15.
m, J= 4.253).
[Where (i) = 3-chloro-4-fluoroaniline; (ii) = Diethylethoxymethylenemalonte (iii) = 3-chloro-4-fluoroanilinomethylene
malonic acid diethyl ester (iv) = ethyl 7-chloro-6-fluoro-4-oxo1, 4-dihydro-quinoline-3-carboxylate]
Scheme 1:
[Where (iv) = ethyl 7-chloro-6-fluoro-4-oxo-1, 4-dihydro-quinoline-3-carboxylate; (v) = ethyl 7-chloro-6-fluoro-4-oxo-
1-R, 4-dihydro-quinoline-3-carboxylate; K2CO3= Potassium carbonate; DMF= N, N, dimethyl formamide].
Scheme 2:

ABDULLAH & SINGH, Orient. J. Chem., Vol. 29(2), 609-615 (2013)
613
RESULTS AND DISCUSSION
Chemistry
The preparation of N-1 substituted
fluoroquinolone derivative I (ethyl 7-chloro-6-fluoro-
4-oxo-1,4-dihydroquinoline-3-carboxylate) is
outlined in scheme 1. 3-chloro-4-fluoroaniline (i)
was treated with diethylethoxymethylenemalonate
(ii) with 1450C heat for 1 hrs. approximate and then
treated with diphenyl ether heated at 2500C
approximate and gives its ethyl 7-chloro-6-fluoro-
4-oxo-1,4-dihydroquinoline-3-carboxylate (iv) over
all yield 80%. The synthetic pathways for
preparation of N-1 substitution in (iv) by treated with
Anhydrous K2CO3, DMF at 1200- 1400C for 5 - 8
hrs. N-1= R (P-toluenesulphonyl chloride, Acetyl
chloride, Benzoyl chloride, Benzyl chloride
respectively.) is outlined in scheme 2. The
physicochemical parameters are recorded are
given in the table 2.
Table 1: Physico-chemical properties of the Derivatives
Derivatives Substituents
R
Melting Point
Yield
(%)
Rf
Solvent System
FI-ETH
FII-SUL
FIII-ATY
H
2500-2530C
2610-2660C
2430-2480C
80
70
67
0.64
0.9
0.82
Ethyl Acetate:Benzene:Acetone5:2:2
Ethyl Acetate:Benzene:Acetone4:2:2
Ethyl Acetate:Benzene:Acetone
5:2.5:2
C7H7ClO2S
C2H3ClO
FIV-BZO
FV-BZY
C7H5ClO
C7H7Cl
2560-2600C
2570-2610C
65
62
0.64
0.48
Ethyl Acetate:Chloroform:Acetone
5:2:2
Ethyl Acetate:Hexane4:1
Table 2:Test Result of Derivatives with Standard Drug Ofloxacin
Organism
Zone of Inhibition (mm)
Derivatives with concentration (µg/ml)
Ofloxacin
(10mg/ml)
FI-ETH
FII-SUL
FIII-ATY
FIV-BZO
FV-BZY
50 100 150 50 100 150 50 100 150 50 100 150 50 100 150
K. Pneumonia 15 21 28 14 22 30 19 24 31 15 19 21 17 19 22 38
B. subtilis
S. aureus
E. coli
26 34 39 25 35 40 23 36 33 20 23 32 29 32 34 42
22 24 40 20 23 36 29 32 38 22 25 35 23 25 27 41
29 30 32 28 29 31 25 30 32 24 29 30 23 26 30 34
Table 3: Antioxidant activity Test Result of Derivatives
Derivatives
DPPH radical scavenging,
IC50 (µg/ ml)a
H2O2 scavenging
IC50 (µg/ ml)b
NO-scavenging
IC50 (µg/ ml)c
FI-ETH
FII-SUL
FIII-ATY
FIV-BZO
FV-BZY
81.69
14.98
68.63
80.32
92.9
287.71
143.51
225.76
245.18
286
79.24
61.73
31.94
68.34
83.46667
Where a IC 50 for Ascorbic acid (standard drug) is 32.0 (µg/ml); b IC 50 for Ascorbic acid (standard drug)
is 67.36 (µg/ml); c IC 50 for Rutin (standard drug) is 15.07 (µg/ml).

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ABDULLAH & SINGH, Orient. J. Chem., Vol. 29(2), 609-615 (2013)
Table 4: Antioxidant activity (Reducing Power Assay) of Derivatives
Concentration Ascorbic acid
FI-ETH
FII-SUL
FIII-ATY
FIV-BZO
FV-BZY
(µg/ml)
(std. drug) Absorbance Absorbance Absorbance AbsorbanceAbsorbance
Absorbance
50
0.029
0.036
0.057
0.078
0.105
0.061
0.070
0.098
0.112
0.129
0.092
0.101
0.129
0.140
0.167
0.074
0.083
0.092
0.116
0.133
0.080
0.089
0.106
0.130
0.147
0.084
0.093
0.121
0.145
0.162
100
150
200
250
In-vitro antibacterial activity
synthesized all derivatives show the hydrogen
All synthesized derivatives are active donating nature, Hydrogen peroxide radical
against gram (+ve) and (-ve) bacterial growth but scavenging activity ability to, nitric oxide assay is
increasing concentration of the derivatives that is Outlined in table 3. and reducing power assay is
increased inhibitory action on bacterial growth.This showed the increasing the absorbance of reaction
inhibition seen on B. subtilis, S. aureus, and E. coli mixture to increase the reducing power, and all
with concentration of 150 µg/ml. as shows in synthesized derivatives show the reducing power.
table 2.
outlined in table 4.
ACKNOWLEDGEMENTS
I special thank to Dr. B. Kundu Deputy
In-vitro antioxidant activity
Antioxidant activity shown is excellent in
all synthesized derivatives, and antioxidant activity
done by four methods DPPH free radical scavenging Director & Head of SAIF CDRI Lucknow (U.P.).
assay is capable of hydrogen donating nature and
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